Breast cancer is one of the most commonly diagnosed female cancers and the second leading cause of cancer death among women [1]. Recently, numerous reports have underscored the important role of cell proliferation rate as a prognostic factor for breast carcinoma. Studies using flow cytometry to measure the DNA content of breast tumor cells show a strong association between a high S-phase fraction and poor prognosis for relapse-free survival in patients with lymph node negative breast cancer [2]. In addition to a high rate of DNA synthesis, mammary cancer cells exhibit extensive DNA damage [3], as compared to non-malignant breast cells. The increased mutation frequency that accompanies the cellular transformation process is postulated to arise from molecular alterations of specific DNA replication and/or repair proteins [4]. Despite the knowledge that a high proliferation activity and increased mutation frequency correlate with breast cancer progression, there is a paucity of information regarding the regulation and precise molecular mechanisms of human breast cell DNA replication.
To date, several mammalian enzymes and proteins have been shown to be required for DNA replication in vitro [5-10]. In particular, the proteins necessary to support SV40 based cell-free DNA synthesis include: DNA polymerase .alpha., DNA primase, DNA polymerase .delta., proliferating cell nuclear antigen (PCNA), replication protein A (RP-A), replication factor C (RF-C), and DNA topoisomerases I and II [11]. As mammalian cell DNA replication represents an intricate yet highly coordinated and efficient process, it follows that the proteins mediating DNA synthesis may be organized into a multiprotein complex. In support of this hypothesis, several reports have described the isolation of large macromolecular complexes of replication essential proteins from extracts of eukaryotic cells [9,11,12].
Our laboratory was the first to isolate and characterize a multiprotein DNA replication complex from human (HeLa) cells and murine (FM3A) mammary carcinoma cells that fully supports origin specific and large T-antigen dependent papovavirus DNA replication in vitro [13-15]. The multiprotein complex was observed to retain its ability to replicate papovavirus DNA after additional purification by anion-exchange chromatography and sucrose or glycerol gradient sedimentation [13-15]. In addition, the integrity of the multiprotein complex was maintained after treatment with salt, detergents, RNase, DNase and electrophoresis through native polyacrylamide gels [15,16]. These results suggest that the association of the proteins with one another is independent of non-specific interactions with other cellular macromolecules.
We report here, for the first time, that breast cancer cells also utilize a multiprotein complex to carry out cellular DNA synthesis, and we now designate this complex the DNA synthesome. We describe the isolation and purification of the DNA synthesome from MDA MB-468 human breast cancer cells and most importantly from human breast tumor cell xenografts, as well as from biopsied human breast tumor tissue. Furthermore, we discuss the results of a forward mutagenesis assay which establish that the DNA synthesome isolated from breast cancer cells and tissue has a lower fidelity for DNA replication than the DNA synthesome isolated from a normal breast cell line. Ultimately, we anticipate that the complete characterization of this DNA synthesome will lead to important new insights into understanding the molecular mechanisms of breast cancer cell DNA replication.